Polyhydroxyalkanoates are polyester-type organic polymers produced by various microorganisms. Actually, PHAs are biodegradable thermoplastic polymers and also producible from renewable resources. Hence, some attempts have been made to industrially produce a PHA as an environmentally friendly material or biocompatible material for various industrial applications.
PHAs consist of units of monomers generally called hydroxyalkanoic acids which are specifically exemplified by 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, other 3-hydroxyalkanoic acids with a longer alkyl chain, and 4-hydroxybutyric acid. The polymer molecules are formed by homopolymerization or copolymerization of these hydroxyalkanoic acids.
Examples of PHAs include poly-3-hydroxybutyric acid (hereinafter abbreviated as P(3HB)) which is a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB); a copolymer of 3HB and 3-hydroxyvaleric acid (hereinafter abbreviated as 3HV) (hereinafter, the copolymer is abbreviated as P(3HB-co-3HV)); and a copolymer of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) (hereinafter, the copolymer is abbreviated as P(3HB-co-3HH)). Other examples include a copolymer of 3HB and 4-hydroxybutyric acid (hereinafter abbreviated as 4HB) (hereinafter, the copolymer is abbreviated as P(3HB-co-4HB)).
The properties of PHAs are dependent on the molecular weight. PHAs having as high a molecular weight as possible are preferred for fiber processing. Thus, the development of techniques to control the molecular weight of PHAs, particularly to increase the molecular weight of PHAs, in a fermentative production process is essential to achieve use of PHAs in industrial applications.
As described below, several techniques for controlling the molecular weight of PHAs have been reported.
Non Patent Literatures 1, 2, and 3 teach a production method for P(3HB) with a weight average molecular weight of higher than 10,000,000 by culturing Escherichia coli cells into which has been introduced Ralstonia eutropha-derived genes involved in PHA synthesis while controlling the pH and glucose concentration. These references show that high molecular weight P(3HB) has better physical properties (e.g., tensile strength and restretchability) which are important for fiber processing or others.
Patent Literature 1 shows that in production of P(3HB) using Escherichia coli cells harboring an expression vector that contains a PHA synthase gene whose expression is under control of an inducible promoter, enzyme expression regulation by varying the amount of inducer enables control of the weight average molecular weight between 780,000 and 4,000,000.
Patent Literature 2 shows that expression of a PHA synthase gene integrated into a bacterial chromosome results in PHAs that have variable molecular weights depending on the integration site. In the case where an Aeromonas caviae-derived PHA synthase gene and genes for supplying substrate monomers were integrated into the Ralstonia eutropha chromosome, PHA copolymers including 3-hydroxyhexanoate and 3-hydroxyoctanoate which have a molecular weight of 400,000 to 10,000,000 were accumulated.
There are also some study reports on control of the molecular weight of P(3HB-co-3HH).
Patent Literature 3 discloses a technique to produce P(3HB-co-3HH) with a weight average molecular weight of 5,100,000 by culturing Ralstonia eutropha cells into which has been introduced Escherichia coli-derived 3-ketoacyl ACP reductase gene (fabG) which encodes an enzyme involved in PHA production, in the presence of a vegetable oil as a carbon source.
As mentioned above, several techniques to control the molecular weight of PHAs, such as control of culture conditions and the activity of PHA synthases and introduction of a gene involved in PHA synthesis, have been reported.
Non Patent Literature 4 shows that C. necator has at least 9 PHA degrading enzymes. Although some study reports on microorganisms with disruptions in any of the genes for these PHA degrading enzymes have been published, what are revealed by these reports are enzyme features and decomposition and utilization of accumulated PHAs, and the influence of disruption on the molecular weight of PHAs is unknown. For example, Non Patent Literature 5 shows that disruption of the phaZ1 gene (which has the base sequence of SEQ ID NO:16, and encodes an amino acid sequence of SEQ ID NO:17) or the phaZ2 gene (which has the base sequence of SEQ ID NO:18, and encodes the amino acid sequence of SEQ ID NO:19) is associated with reduced decomposition of P(3HB) accumulated in C. necator cells.
As for the phaZd gene (phaZ6 gene), which is a member of the PHA degrading enzyme gene family, Non Patent Literature 6 shows that disruption of this gene does not affect decomposition and utilization of PHAs. Thus, how the phaZ6 gene works in cells is unknown.